A protein exhibiting an antifreeze effect on aqueous solutions is generally referred to as an antifreeze protein (AFP). Various antifreeze protein have been found in living organisms such as fish, insects, plants, fungi and bacteria which typically have adaptability to low temperature environments. It is know that all antifreeze proteins originated from fish and plants allow an ice nucleus to grow to an ice crystal having a bi-pyramid shape, just like a pair of triangular pyramids joined together at their bottom surfaces. This mechanism is explained as follows. Under usual conditions, upon generation of an ice nucleus in an aqueous solution, an ice crystal first grows to have a flat hexagonal plate shape. In this case, the ice crystal has a growth rate in the vertical direction 100 times lower than that in the plate plane direction. In contrast, when an antifreeze protein is present in the aqueous solution, the ice crystal gradually grows to the bi-pyramid-shaped ice crystal under restraint on its growth in the plate plane direction in such manner that a plate-shaped body is initially formed to provide a base plane, and a plurality of small plate-shaped bodies are sequentially piled up on both sides of the plate-shaped body in the vertical direction with respect to the base plane.
An antifreeze protein dissolved in an aqueous solution brings about an antifreeze effect on the aqueous solution, such as 1) thermal hysteresis, 2) ice-recrystallization inhibition, and 3) ice crystal shape control. While the water freezing point is generally equal to the ice melting point, an aqueous solution containing an antifreeze protein has a depressed water freezing point because the protein is bonded to an ice crystal to be formed. This phenomenon is referred to as “thermal hysteresis,” and the difference between the melting point of the ice formed therein and the water freezing point is defined as “depression of freezing point.” A greater depression of freezing point means a greater antifreeze effect. The ice crystal formed therein grows while absorbing water generated by sublimation or partial melting at a relatively high temperature of −10° C. or more. Inhibition of this phenomenon is defined as “ice-recrystallization inhibition.” A higher ice-recrystallization-inhibition activity means a higher antifreeze effect. By taking advantage of the above properties of the antifreeze protein, the antifreeze protein has been proposed for use as an additive for ice cream apt to deteriorate in its flavor or taste due to attachment/recrystallization of water molecules in ambient air caused by cold insulation, or as a cryopreservative for cells and organs. The antifreeze protein is also expected to function as an effective additive for eliminating clogging of pipelines due to ice recrystallization in a system using ice slurry, such as cryogenic supply systems or cryogenic storage systems.
However, it is difficult to assure a large, stable supply of most of the known antifreeze proteins originated from plants and animals. Therefore, recombinant gene technology has been used to produce some antifreeze proteins originated from fish or insects, and to make the proteins more stable. However, antifreeze proteins produced by recombinant methods have not been used in foods for human consumption because of consumer opposition to gene-altered products. While some antifreeze proteins have been successfully purified from bacteria, they are not suitable for human consumption due to the properties related to their bacterial origin, and due to insufficient stability. While it has been reported that some antifreeze proteins exist in basidiomycetes, which are widely utilized for human consumption, no antifreeze protein has been isolated/purified therefrom.
While various attempts have heretofore been made to put natural antifreeze proteins mainly originated from plants or fish to practical use as a quality improving agent for frozen foods such as ice cream, as a cryopreservative for cells, and as an additive for cryogenic supply systems or cryogenic storage systems, no practical application has been achieved due to instability in activity of the conventional antifreeze proteins and the resulting need for using them in large quantities to bring about desired functions in view of their poor stability.